The discovery that molybdenum functions in many molybdoenzymes of animal, plant and bacterial origin as a complex with a unique, sulfur-containing compound termed molybdopterin and the identification of a number of severely ill patients who lack active molybdenum cofactor emphasize the need for further investigations into the metabolic processes involved in utilization of this trace element. The overall objective of this project will be to understand how the molybdenum cofactor is handled in human tissues: where it comes from, how and in what form it gets to where it is utilized and finally how it is degraded or processed for excretion. If all or part of the molybdopterin requirement is satisfied by de novo synthesis, the pathway and site of synthesis need to be determined. If all or part of the cofactor is obtained from the diet, it is important to establish the chemical nature of the nutrient(s), as well as how it is taken up and transported. The specific goals for this project period will be to evaluate three hypotheses with regard to the metabolic origins of molybdopterin: 1. Biosynthesis of molybdopterin in animal tissues. Radioactive guanine or guanosine will serve as the substrate to test for in vivo transfer of label to molybdopterin. The involvement of GTP cyclohydrolase I in molybdopterin synthesis will be assessed using labeled dihydroneopterin triphosphate as substrate for in vitro studies. The possible role of a GTP cyclohydrolase II will be addressed by establishing the presence or absence of such an activity in animal tissues and, if it is shown to be present, by monitoring in vitro conversion of its reaction product to molybdopterin. 2. Dietary origins of molybdopterin. Levels of molybdenum and molybdopterin or related molecules will be quantitated in selected foodstuffs, solutions for total parenteral nutrition, and tissue culture media. The feasibility of preparing sulfur labeled nitrate reductase for feeding experiments will be explored. 3. Folic acid as possible precursor of molybdopterin. The effects of methotrexate treatment and experimental folate deficiency will be tested in several systems. These studies should ultimately provide a rational basis for design of compounds that may alleviate symptoms of molybdenum cofactor deficiency and may have benefit also to individuals on total parenteral nutrition and the recently discovered population of patients who suffer from sulfite-sensitive asthma.